Antibodies Targeted to Fungal Cell Wall Polysaccharides

ABSTRACT

A compound comprising one or more polysaccharide moieties each independently represented by the formula β(1→4)-[GlcNH-R]n-2,5-anhydromannose, wherein n is a positive integer from 3 to 500, and R is H or an acyl group, is described. The compound can be manufactured by (a) reacting chitosan with an acylating agent sufficient to partially N-acylate the chitosan, yielding a modified chitin/chitosan mixed polymer; and (b) reacting the modified chitin/chitosan mixed polymer with a deaminating agent to cleave the mixed polymer at the unacylated chitosan moieties. The compound can be used to immunize against fungal infection. Antibodies specific to the compound, and the use of such antibodies to protect against fungal infection are also described.

BACKGROUND OF THE INVENTION

A dramatic rise in the incidence of invasive fungal disease in recentyears, as well as the emergence of drug resistant and previously rarefungal species, has highlighted the need for broadly effective newtherapeutic and prophylactic antifungal treatment strategies (3-5). Theincreased incidence of invasive fungal infection is partly attributableto an increase in the immunocompromised patient population, owing to thegrowing number of patients with disease associated acquiredimmunodeficiencies, those in critical care units, patients undergoingsurgery or immunosuppressive treatment, and those receiving organ orcellular transplant therapies. Importantly, the risk factors thatpredispose individuals to invasive fungal disease do not preclude thepossibility of mounting an effective immune response and respondingfavorably to immunotherapy (6, 7), giving promise to the development ofeffective vaccine or immunotherapy based approaches to meet thisunderserved medical need.

The two most prevalent pathogenic fungi affecting humans, Aspergillusand Candida spp., account for an estimated 8-10% of all health careacquired infections, with an attributable mortality of 30-40% (5, 8).Candida species are the fourth leading cause of nosocomial sepsis casesin the US and the rising incidence of invasive fungal disease from allpathogenic fungi represents a significant healthcare burden worldwide.Excess healthcare costs due to increased length of stay and treatment ofhospital acquired fungal infections are in the range of US$1 thousandmillion annually in the US alone. Furthermore, comprehensive antifungalsusceptibility testing of clinical isolates has made it evident that,despite advances in safe and effective antifungal drugs, all classes ofcurrently available antifungal agents are subject to the emergence ofresistant strains (5). Cryptococcal meningitis, an infection with thefungus Cryptococcus also known as cryptococcosis, is a very seriousopportunistic infection among people with advanced HIV/AIDS.Cryptococcosis is not contagious, meaning it cannot spread fromperson-to-person. Cryptococcal meningitis specifically occurs afterCryptococcus has spread from the lungs to the brain. A global problem,worldwide, approximately 1 million new cases of cryptococcal meningitisoccur each year, resulting in 625,000 deaths. Most cases areopportunistic infections that occur among people with HIV/AIDS. Althoughthe widespread availability of antiretroviral therapy (ART) in developedcountries has helped reduce cryptococcal infections in these areas, itis still a major problem in developing countries where access tohealthcare is limited. Throughout much of sub-Saharan Africa, forexample, Cryptococcus is now the most common cause of adult meningitis.Cryptococcal meningitis is one of the leading causes of death inHIV/AIDS patients; in sub-Saharan Africa, it may kill as many peopleeach year as tuberculosis.(24).

These significant challenges of combating fungal disease point to thecritical need for a highly effective pan-fungal vaccine as a valuablecomponent of the anti-fungal arsenal. To date, two fungal cell wallcarbohydrate components, β-mannan and β-glucan, have been explored astargets for anti-fungal vaccination. Conjugate vaccines composed ofeither linear β-(1→3)-glucan or β-(1→2)-mannotriose have been shown toconfer protection against fungal disease, with efficacy in both activeand passive immunization (9-11). In animal models of fungal disease, theβ-glucan vaccine proved effective against C. albicans, A. fumigatus, andC. neoformans, validating the possibility of successful vaccinationagainst multiple, disparate fungal pathogens (9, 12). Nevertheless, theproduction of effective vaccine responses requires careful considerationof the fine structure of the target antigens, as there is mountingevidence that one mechanism of immune evasion employed by fungalpathogens is the expression of immunodominant epitopes that inducenon-protective or inhibitory antibody responses. These decoy epitopesablate the efficacy of responses toward protective epitopes. Forexample, vaccines composed of linear β-(1→3)-glucan epitopes produceprotective responses, while vaccines composed of β-(1→3)-glucans withβ-(1→6)-glucan branches produce antibodies to both structures but do notconfer protection against fungal disease (13, 14).

Moreover, the fundamental utility of the vaccine is dependent on theuniversality of target antigens. For example, the β-(1→2)-mannotrioseepitope does not appear in all Candida species and the vaccine antigenemploying this epitope relies on protective peptide epitopes to expandits utility (10). Considering the limited distribution of some cell wallcarbohydrate epitopes and in view of the mechanisms employed by fungalpathogens to avoid productive immune responses to cell wall components,there is a need for a universally effective fungal vaccine. Thisinvention is designed to target highly conserved fungal cell wallcarbohydrate epitopes in order to provide a pan-fungal vaccine.

Chitin has not been examined as an antifungal vaccine target, largelyfor reasons related to its highly insoluble nature. Methods available inthe art for degrading chitin into soluble fragments are notstoichiometrically controlled and it is thus difficult to modulate thedegree of depolymerization.

SUMMARY OF THE INVENTION

This invention provides a compound comprising one or more polysaccharidemoieties each independently represented by the formulaβ(1→4)-[GlcNH-R]_(n)-2,5-anhydromannose, wherein n is a positive integerfrom 3 to 500, and R is H or an acyl group.

This invention also provides a process for manufacturing a compoundrepresented by the formula β(1→4)-[GlcNH-R]_(n)-2,5-anhydromannose,wherein n is a positive integer from 3 to 500, comprising:

(a) reacting chitosan with an amount of an acylating agent sufficient topartially N-acylate the chitosan, yielding a modified chitin/chitosanmixed polymer;(b) reacting the modified chitin/chitosan mixed polymer with adeaminating agent to cleave the mixed polymer at the unacylated chitosanmoieties, yielding the compound of formulaβ(1→4)-[GlcNH-R]_(n)-2,5-anhydromannose.

This invention provides a method of immunizing a mammalian subjectagainst a fungal infection or pathogen, comprising administering to thesubject an immunogenic amount of the compound or a compositioncontaining it. This invention provides a method of stimulating an immuneresponse in a mammalian subject against a fungal pathogen, comprisingadministering to the subject an immunogenic amount of the compound or acomposition containing it. This invention also provides an antibodyspecific to the compound described above. And it provides a method ofprotecting a mammalian subject against a fungal infection or pathogen,comprising administering the antibody to the subject in an amounteffective to protect the subject against the fungal infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Reaction scheme for the preparation of modified chitinfragments.

Reaction scheme for the one pot preparation of modified chitin fragmentsfrom chitosan.

FIG. 2: Analysis of modified chitin conjugation to tetanus toxoid.

Coomassie Blue stained SDS-PAGE gel showing conjugation of modifiedchitin fragments to tetanus toxoid (TT) protein.

FIG. 3: Dose and immunization schedule modified chitin-TT conjugate.

Chart showing dose and immunization schedule for administration ofmodified chitin-TT conjugate vaccine to Balb/C mice.

FIG. 4: Immunogenicity of modified chitin-TT conjugate vaccine in Balb/Cmice.

Line plot showing immunogenicity of modified chitin-TT vaccine conjugatein Balb/C mice. The screening antigen was modified chitin cross linkedto human serum albumin (HSA).

FIG. 5A: Specificity of immune response in Balb/C mice immunized withmodified chitin-TT vaccine conjugate.

Line plot comparing inhibition of vaccine serum binding to modifiedchitin-HSA screening antigen.

FIG. 5B: Specificity of immune response in Balb/C mice immunized withmodified chitin-TT vaccine conjugate.

Bar graph showing lack of cross reactivity of vaccine sera towardmultiple GlcNAc containing glyconjugates.

FIG. 5C: Specificity of immune response in Balb/C mice immunized withmodified chitin-TT vaccine conjugate.

Bar graph showing reactivity of vaccine sera toward chitin/chitosanpolysaccharides. The graph shows that antibodies reactive towardmodified chitin can be depleted by adsorption on particulate chitin orchitosan. In addition, soluble extracts from chitin or chitosan inhibitbinding of vaccine sera to modified chitin-HSA.

FIG. 5D: Specificity of immune response in Balb/C mice immunized withmodified chitin-TT vaccine conjugate.

Bar graph showing reactivity of vaccine sera toward chitin/chitosanpolysaccharides.

The graph shows that binding of an irrelevant antibody (anti-HA) to itsepitope is unaffected by the same treatments.

FIG. 6A: Binding of modified chitin-TT conjugate vaccine inducedantibodies to whole Candida albicans fungi.

Bar graph showing that serum antibodies from mock (PBS) and modifiedchitin-TT immunized Balb/C mice bind to whole C. albicans. Reactivity isspecifically competed away with modified chitin fragments.

FIG. 6B: Binding of modified chitin-TT conjugate vaccine inducedantibodies to whole Candida albicans fungi.

Bar graph showing that antigen affinity purified chitin reactiveantibodies bind to whole C. albicans.

FIG. 7A: Morphology of Candida albicans yeast cells grown under variousconditions Yeast cells grown at 30 C in YPD medium o/n.

FIG. 7B: Morphology of Candida albicans yeast cells grown under variousconditions Intermediate filaments cells grown at 37 C in YPD medium andserum for 150 min.

FIG. 7C: Morphology of Candida albicans yeast cells grown under variousconditions Filaments grown into mycelia at 37 C in YPD medium and serumfor 300 min.

FIG. 8A: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Assay control (gating strategy) for antibody binding to yeast cells.

FIG. 8B: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Assay controls (unstained control) for antibody binding to yeast cells.

FIG. 8C: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Positive assay control (rabbit polyclonal antibody to candida) forantibody binding to yeast cells (1:50 dilution).

FIG. 8D: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Positive assay control (rabbit polyclonal antibody to candida) forantibody binding to yeast cells (1:100 dilution).

FIG. 8E: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Positive assays control (rabbit polyclonal antibody to candida) forantibody binding to yeast cells 1:200 dilution.

FIG. 9A: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Tetanus toxoid antibody controls binding to yeast cells.

FIG. 9B: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Tetanus toxoid antibody controls binding to yeast cells.

FIG. 10A: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 30 C in YPD mediumo/n.

FIG. 10B: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells grown at 30 C in YPDmedium o/n.

FIG. 10C: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 30 C in YPD mediumo/n.

FIG. 10D: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells grown at 30 C in YPDmedium o/n.

FIG. 10E: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 30 C in YPD mediumo/n.

FIG. 10F: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells grown at 30 C in YPDmedium o/n.

FIG. 11A: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 30 C in YPD mediumo/n (1:10 dilution).

FIG. 11B: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 30 C in YPD mediumo/n (1:50 dilution).

FIG. 11C: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 37 C in YPD mediumand serum for 150 min (1:10 dilution).

FIG. 11D: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 37 C in YPD mediumand serum for 150 min (1:50 dilution).

FIG. 11E: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 37 C in YPD mediumand serum for 300 min (1:10 dilution).

FIG. 11F: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Laminarin antibody binding to yeast cells grown at 37 C in YPD mediumand serum for 300 min (1:50 dilution).

FIG. 12A: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells (mycelia) when grown at30 C in YPD medium o/n (1:10 dilution).

FIG. 12B: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells (mycelia) when grown at30 C in YPD medium o/n (1:50 dilution).

FIG. 12C: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells (mycelia) when grown at37 C in YPD medium and serum for 150 min (1:10 dilution).

FIG. 12D: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells (mycelia) when grown at37 C in YPD medium and serum for 150 min (1:50 dilution).

FIG. 12E: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells (mycelia) when grown at37 C in YPD medium and serum for 300 min (1:10 dilution).

FIG. 12F: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Candida albicans by flow cytometry.

Modified chitin antibody binding to yeast cells (mycelia) when grown at37 C in YPD medium and serum for 300 min (1:50 dilution).

FIG. 13A: Modified chitin-TT vaccine mediated protection from a lethalchallenge of C. albicans

Mice (Balb/C) were immunized with a modified chitin-TT vaccine andsubsequently challenged with a lethal dose of live C. albicans. Survivalwas monitored for 36 days after fungal challenge.

FIG. 13B: Modified chitin-TT vaccine mediated protection from a lethalchallenge of C. albicans

Mice (CD1) were immunized with a modified chitin-TT and laminarin-TTconjugate vaccines and subsequently challenged with a lethal dose oflive C. albicans. Survival was monitored for 28 days after fungalchallenge.

FIG. 14A: Immunoreactivity by ELISA of normal human sera with chitin;modified chitin and laminarin

Reactivity of normal human sera (IgG gamma) on a modified chitin-HSAcoated plate. All sera react significantly with modified chitinsuggesting the presence of naturally acquired chitin-specific antibodiesin human through either exposure to fungi or other chitin containingforeign antigens.

FIG. 14B: Immunoreactivity by ELISA of normal human sera (NHS) withchitin; modified chitin and laminarin

Antibody specificity of binding of a high-titer NHS to modifiedchitin-HSA coated plate by competitive inhibition with variousinhibitors. NHS specificity towards modified chitin is the highest,followed by the small chitin oligosaccharide inhibitors (DP5 and DP3).

FIG. 14C: Immunoreactivity by ELISA of normal human sera with chitin;modified chitin and laminarin

Antibody specificity of binding of a high-titer NHS to Chitohexaose-HSAcoated plate by competitive inhibition with chitin oligosaccharides ofincreasing DP. There is an increase inhibition with increasing size ofthe chitin oligosaccharides from DP2 to DP6, suggesting that these humanantibodies recognize a conformational epitope of chitin with a minimumsize of an hexasaccharide.

FIG. 14D: Immunoreactivity by ELISA of normal human sera with chitin;modified chitin and laminarin

Reactivity of normal human sera (IgG gamma) on a laminarin-HSA coatedplate. NHS recognize laminarin (beta glucan) antigen although not to thesame degree as modified chitin.

FIG. 15A: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Cryptococcus neoformans type A (H99) by flowcytometry. Assays control for antibody binding to yeast cells. Thispanel shows GCMP-TT Negative Control

FIG. 15B: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Cryptococcus neoformans type A (H99) by flowcytometry. Assays control for antibody binding to yeast cells. Thispanels shows Laminarin-TT

FIG. 15C: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Cryptococcus neoformans type A (H99) by flowcytometry. Assays control for antibody binding to yeast cells. Thispanel shows Positive Control: Mouse monoclonal antibody to Cryptococcus.

FIG. 15D: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Cryptococcus neoformans type A (H99) by flowcytometry. Assays control for antibody binding to yeast cells. Thispanel shows mChitin-25%-TT.

FIG. 15E: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Cryptococcus neoformans type A (H99) by flowcytometry. Assays control for antibody binding to yeast cells. Thispanel shows mChitin-75%-TT.

FIG. 15F: Binding of modified chitin and laminarin vaccine-inducedantibodies to whole Cryptococcus neoformans type A (H99) by flowcytometry. Assays control for antibody binding to yeast cells. Thispanel shows mChitin-85%-TT.

DETAILED DESCRIPTION OF THE INVENTION

This invention provide s compound comprising one or more polysaccharidemoieties each independently represented by the formulaβ(1→4)-[GlcNH-R]_(n)-2,5-anhydromannose, wherein n is a positive integerfrom 3 to 500, and R is H or an acyl group. In more specific embodimentsof this invention, n is a positive integer from 3 to 100, or from 6 to50. In an embodiment the acyl group R is an acetyl. In anotherembodiment at least 30% of the acyl groups in the compound are acetyl.

The technical field is prevention, treatment, and detection of fungalinfections. Specifically, vaccines or immunotherapeutics that targetcarbohydrate components of the fungal cell wall can provide treatmentfor disseminated or locally invasive fungal infections. The diseaseindications are numerous, including, but not limited to those caused byhuman pathogenic forms of Candida, Aspergillus, and Cryptococcusspecies. Additionally, the ability to generate an antibody response tothe conserved carbohydrate components of these pathogenic fungi couldlead to the development of diagnostic reagents for detection of thesefungal agents in patient biological samples (eg. plasma, serum, or otherbodily fluids, as well as tissue sections, etc.). Finally, since Th2type inflammatory responses to chitin have been implicated in allergicasthma and other allergic conditions (2), a chitin based vaccine thatresults in a shift toward a Th1 type immune response may ameliorate thesymptoms of allergen induced inflammation.

Another fundamental cell wall carbohydrate of interest is the surfaceα-1,3-glucan. Successful infection by fungal pathogens depends onsubversion of host immune mechanisms that detect conserved cell wallcomponents such as beta-glucans. A less common polysaccharide,α-(1,3)-glucan, is a cell wall constituent of most fungal respiratorypathogens and has been correlated with pathogenicity or linked directlyto virulence. However, the precise mechanism by which α-(1,3)-glucanpromotes fungal virulence is unknown. α-(1,3)-glucan is present in theoutermost layer of the Histoplasma capsulatum yeast cell wall andcontributes to pathogenesis by concealing immunostimulatory beta-glucansfrom detection by host phagocytic cells. Production of proinflammatoryTNFalpha by phagocytes was suppressed either by the presence of theα-(1,3)-glucan layer on yeast cells or by RNA interference baseddepletion of the host beta-glucan receptor dectin-1. Rappleye et al.have functionally defined key molecular components influencing theinitial host-pathogen interaction in histoplasmosis and have revealed animportant mechanism by which H. capsulatum thwarts the host immunesystem. Furthermore, they propose that the degree of this evasioncontributes to the difference in pathogenic potential between dimorphicfungal pathogens and opportunistic fungi (25). Another study suggestedthe relevance of cell wall α-1,3-glucan for fungal infection. Becausemany fungal species could potentially utilize the same or similarstealth infection strategies, targeting the fungal α-1,3-glucan, forexample, by conferring α-1,3-glucanase activity to crop plants orapplying an inhibitor of the fungal α-1,3-glucan synthase might providea versatile strategy for the prevention of a wide variety of fungaldiseases in important crops. Although the detailed mechanism is stillremains to be solved, the fact is that the removal of the surfaceα-1,3-glucan rapidly activates the defenses responses of the host plantagainst the fungal pathogens prior to the fungal invasion, resulting inthe inhibition of pathogen infection (26). Yeast cell walls are criticalfor maintaining cell integrity, particularly in the face of challengessuch as growth in mammalian hosts. The pathogenic fungus Cryptococcusneoformans additionally anchors its polysaccharide capsule to the cellsurface via α(1-3) glucan in the wall. Cryptococcal cells disrupted intheir alpha glucan synthase gene were sensitive to stresses, includingtemperature, and showed difficulty dividing. These cells lacked surfacecapsule, although they continued to shed capsule material into theenvironment. Electron microscopy showed that the alpha glucan that isusually localized to the outer portion of the cell wall was absent, theouter region of the wall was highly disorganized, and the inner regionwas hypertrophic. Analysis of cell wall composition demonstratedcomplete loss of alpha glucan accompanied by a compensatory increase inchitin/chitosan and a redistribution of beta glucan between cell wallfractions. The mutants were unable to grow in a mouse model ofinfection, but caused death in nematodes. These studies integratemorphological and biochemical investigations of the role of alpha glucanin the cryptococcal cell wall (27).

Thus a vaccine comprising chitin/chitosan and one or more glucanantigens, for example glucan antigens containing homopolymers of α-1,3linkagers, β-1,3 linkages or both, will target highly conserved fungalcell wall epitopes and provide a pan-fungal vaccine.

This invention provides anti-fungal conjugate vaccines that targetconserved carbohydrate cell wall components that are common structuralelements across multiple phyla of pathogenic fungi. The composition ofthese vaccines can include one or more epitopes consisting ofoligosaccharides or polysaccharides derived from chitin, alone or incombination with β-mannan or glucan epitopes. The carbohydratecomponents may be conjugated to an appropriate immunogenic proteincarrier, such as tetanus toxoid, diptheria toxoid, or specific proteinvirulence factors present on the fungal cell surface. In an embodimentof this invention the carrier protein is covalently linked directly tothe one or more polysaccharide moieties at the anhydromannose group ofeach of the polysaccharide moieties. In an embodiment, the carrierprotein is covalently linked to the one or more polysaccharide moietiesvia one or more immunogenic scaffold moieties. Any conventional scaffoldmoiety can be used, examples of which include a polysaccharide, apeptidoglycan, a polypeptide, and a polyglycerol. The scaffold can belinear or dendrimeric. Envisioned multicomponent vaccines can include,but are not limited to, a simple mixture of chitin, β-mannan and/orglucan epitopes (linear α-1,3- and/or β-1,3-glucans) or derivativesconjugated individually to a carrier protein or immunogenic scaffold ofchoice.

In one embodiment, the β-mannan can be a β-1,2-mannose tetrasaccharide.In the simplest embodiment of a β-1,2 mannose tetrasaccharide/scaffoldcomplex, the oligosaccharides can be reacted with a modified chitinpolysaccharide, to produce a multivalent molecule. The β-1,2 mannosetetrasaccharide can be chemically synthesized (23). The condensation ofthe oligosaccharide can be accomplished by any of numerous methodsavailable in the art for reaction between the reducing end of theoligosaccharide with the free amino groups of the modified chitin or viaa spacer arm equipped with a squarate group (23). In addition, theinvention envisions a further step of conjugating the scaffoldedoligosaccharide/polysaccharide to a suitable carrier protein. Thisconjugation may be achieved by a number of means available to apractitioner skilled in the art. For example, the scaffold complex canbe conjugated directly by reductive amination, or indirectly byemploying a suitable cross-linker containing a spacer length suitable torelieve steric hindrance between the carbohydrate construct and thecarrier protein. In yet another embodiment of the invention, adendrimeric polysaccharide scaffold can be prepared by first reactingthe scaffold component with a multifunctional cross-linking reagent,such as Tris-succinimidyl-aminotriacetate (TSAT). This reaction wouldyield a multivalent array of available scaffolds, in a dendrimer likepattern, for subsequent condensation of the β-1,2 mannoseoligosaccharides component.

Alternatively, the different carbohydrate components can be chemicallycross linked, either directly or indirectly. For example, thecarbohydrates can be joined by linking them together on a commonscaffold matrix, such as a dendrimeric substrate, or by co-conjugationto a protein carrier. Yet another embodiment is to directly scaffold thecomponents together by creating chemical cross links between thecarbohydrate components and subsequently conjugating the scaffoldedpolysaccharide matrix to an immunogenic protein or peptide carrier.These chemical cross links can be achieved by any number of meansavailable to a practioner skilled in the art, including, but not limitedto reductive amination or the use of heterobifunctional orhomobifunctional chemical cross linking reagents.

This invention provide a composition comprising the compound asdescribed above, wherein for at least 80% of molecules of the compoundin the composition n has a value from 6 to 50. In an alternativeembodiment, this invention provides a composition comprising moleculesof the compound as described above, wherein the mean value of n is from10 to 50.

Methods to produce chitin or chitosan derived fragments for use in ananti-fungal vaccine were tested. Numerous strategies were tested, undervarious conditions, based in part on methods available in the literature(15-18). Approaches included methods to directly obtain chitinoligosaccharides, such as partial acid hydrolysis of chitin. Variablestested included time, temperature, and scale of reaction. In all testedmethods, solubility was a major problem and yield of oligosaccharides ofthe desired size was in the disappointing range of 1% or less,consistent with published yields, but undesirable for efficientproduction of a vaccine antigen. Also tested were various conditions,based on published methods, to achieve limited nitrous deamination ofchitosan oligosaccharides or polysaccharides, which deaminates freeamino groups with concomitant glycosidic bond cleavage (polymerfragmentation) at the deaminated residue. The variables tested fornitrous deamination included varying the mole fraction of nitrous acidand starting with chitosan of varying size and degree of acetylation. Inall of these cases, the nitrous acid treatment solubilized the chitosansuspensions, but the reaction was difficult to control and once again,the yield of oligosaccharides of the desired size was low. Finally, inan attempt to produce a partially de-N-acetylated chitin as the startingmaterial for the nitrous deamination reaction, chitin was treated withNaOH to effect a limited de-N-acetylation prior to nitrous deamination.This treatment only produced chitin oligosaccharides of very lowmolecular weight.

Failing to obtain chitin derived fragments of the desired size inacceptable yield with the existing methods according to the art, anotherstrategy for obtaining the desired product was devised, which was topartially re-N-acylate (e.g., re-N-acetylate) chitosan in order toproduce a chitin/chitosan mixed polymer with a limited number ofglucosamine residues containing free amino groups. Because thesubsequent nitrous deamination reaction only occurs at glucosamineresidues containing free amino groups, the N-acyl glucosamine (e.g.,N-acetyl glucosamine) residues would be resistant to cleavage. Since theacylation (e.g., acetylation) reaction is not catalytic in nature,whether the reaction could be controlled by adjusting the mole fractionof acetylating reagent present in the reaction was tested. By adjustingthe quantity of acylating (e.g., acetylating) reagent, it was found thatone could directly control the size of the chitin derived fragments inthe subsequent nitrous deamination reaction. Furthermore, the tworeactions leading from chitosan to modified chitin fragments could beperformed as a one pot reaction, consisting of a solid phase acylation(e.g., acetylation) reaction, followed by acidification and nitrousdeamination to yield modified chitin fragments. Any convention acylatingagent can be used, for example acetic anhydride or acetyl chloride, bothof which are acetylating agents, or N-propionic anhydride or propionicchloride, which are propionylating agents.

A major impediment to employing chitin in a vaccine formulation is itshighly insoluble nature. Methods available in the art for degradingchitin into soluble fragments are not stoichiometrically controlled andit is thus difficult to modulate the degree of depolymerization. The aimwas to efficiently produce chitin derived fragments of sufficient sizeto induce vaccine responses against native chitin polymers in the fungalcell wall, while also meeting the potentially competing criteria thatthe fragments be soluble or sufficiently uniform that they are suitablefor formulation as an injectable vaccine. The non-limiting examplesbelow demonstrate how success was achieved in meeting these criteria ina one pot reaction. By employing a first step of partialre-N-acetylation of chitosan in a stoichiometrically limited reaction,it was possible to subsequently fragment the modified polymer by nitrousdeamination, in a controlled fashion (Example 1 and FIG. 1).

This invention provides a method of immunizing a mammalian subjectagainst a fungal infection, comprising administering to the subject animmunogenic amount of a compound or composition as described above. Thesubject can be a human or non-human animal.

In order to improve the immunogenicity of the carbohydrate antigen andto promote a T-cell dependent memory response, the modified chitinfragment was conjugated to tetanus toxoid. Tetanus toxoid is anon-limiting example of an immunogenic protein that contains multipleT-helper epitopes which make it suitable for use as a protein carrierfor carbohydrate conjugate vaccines. Example 2 and FIG. 2 demonstrate anon-limiting example of a method for conjugation of the aldehydecontaining chitin fragments to tetanus toxoid via reductive amination.Example 3 and FIG. 3 provide a typical example of an immunizationstrategy to induce vaccine responses in test animals.

Evaluation of antibody responses in Balb/C mice immunized with themodified chitin vaccine, as outlined here, demonstrates that the vaccineproduces robust and specific immune responses toward both the immunizingantigen (Example 4 and FIG. 4) and toward whole C. albicans fungi(Example 6 and FIGS. 6A and 6B). The mock (PBS) immunized animalsexhibit a basal level of binding to whole C. albicans (Example 6A andFIG. 6A), consistent with prior fungal exposure. Immunization with themodified chitin vaccine markedly elevated the titer of serum antibodiesthat recognize C. albicans, indicating an enhancement of the adaptiveresponse to the fungus. Binding of C. albicans was substantiallyinhibited with modified chitin fragments, demonstrating that asignificant portion of the serum antibodies recognize chitin and,importantly, that the antibodies recognize chitin in the native fungus.Antigen inhibition experiments verified the specificity of the vaccineresponse toward the immunizing antigen (Example and FIG. 5A).Furthermore, the induced antibodies showed no detectable crossreactivity with multiple GlcNAc containing glycoconjugates that arepresent in mammals (Example 5B and FIG. 5B). These negative specificitycontrols included O-linked GlcNAc-Serine, a major intracellular glycan;ovalbumin and fetuin, which both contain many different N- and O-linkedcarbohydrate antigens; hyaluronic acid (HA), which is a major componentof the extracellular matrix; and crude serum, which contains a multitudeof glycoproteins. When the vaccine sera was preadsorbed on a suspensionof chitin or chitosan particles, or incubated with solublechitin/chitosan, binding to the modified chitin antigen was dramaticallyinhibited (Example 5C and FIGS. 5C and 5D), further demonstrating thespecificity of the vaccine response. Antigen affinity purification ofthe vaccine induced serum antibodies show that the chitin specificfraction is capable of binding whole C. albicans. These data provide aconcrete example of a vaccine directed toward a conserved and essentialcomponent of the fungal cell wall, namely chitin, which is present inall known pathogenic fungi.

Analysis of the binding of modified chitin vaccine induced antibodies toC. albicans yeast cells by flow cytometry indicate that the modifiedChitin antibodies stained positively candida cells with about 25% oflive population binding at 1:10 dilution (Example 6C and FIGS. 10B, 10Dand 10F) further supporting the binding data detailed in Example 6. Hightiter laminarin (β-glucan) antibodies generated with a laminarin-TTconjugate also stained positively C. albicans (FIGS. 10A, 10C and 10E).These data suggest that both carbohydrates are exposed at the surface ofthe fungus.

The binding of modified chitin antibodies as well as beta-glucanantibodies at various stages of candida growth in culture was alsoexamined by flow cytometry. Yeast cells (following overnight incubationat 300 C), and mycelial (filament) forms (following incubation at 370 Cfor 150 minutes and 300 minutes respectively, were obtained by culturein YPD medium containing serum (FIGS. 7A-C).

The flow cytometry staining results indicate that the binding of thevaccine-induced laminarin antibodies to candida cells is highest onyeast cells and decreases as the cells differentiate to the morevirulent filament forms of the fungus (FIGS. 11A through 11F) whereasthe binding of modified chitin induced antibodies are not affected bythe differentiation stages of the fungus indicating their consistentantigenic expression throughout the invasive process (FIGS. 12A through12F). These results are important for the development of an effectivepan-fungal vaccine formulation in that it stresses the need toincorporate more than one cell wall carbohydrate component in thevaccine formulation for an optimum efficacy throughout the fungalinvasive process.

Analysis of the binding of modified chitin vaccine induced antibodies toCryptococcus neoformans type A (H99) yeast cells by flow cytometryindicate that the modified Chitin antibodies stained positivelycryptococcal cells with about 31%, 47% and 60% of live populationbinding at 1:10 dilution with the 25%, 75% and 85% modified chitin-TTconjugate antibodies respectively (Example 6 and FIGS. 15D, 15E and 15)further supporting the binding data detailed in Example 6. High titerlaminarin (β-glucan) antibodies generated with a laminarin-TT conjugatealso stained positively Cryptococcus neoformans (FIGS. 15A, 15B and15C). As for Candida albicans these data suggest that both carbohydratesare exposed at the surface of the cryptococcal fungus.

In a preliminary example of protection against a lethal challenge with apathogenic fungus, Balb/C and CD1 mice that received a single cell wallcomponent vaccine comprising a modified chitin-tetanus toxoid conjugate,showed partial protection against a 100% lethal dose of live C. albicans(Example 7 and FIGS. 13A and 13B). In a repeat lethal challengeexperiment, the same modified chitin-tetanus toxoid conjugate vaccinedisplayed similar partial protection against live C. albicans, albeitgreater protection than the a laminarin (β-glucan)-tetanus toxoidconjugate or an chitohexaose-tetanus toxoid conjugate (FIG. 13B). Theseresults will be extended and further embodiments of the invention, suchas vaccines comprising multiple cell wall components (Example 9 andFIGS. 8A through 12F) will be examined for efficacy in similar in vivolethal challenge models employing, for example, C. albicans, A.fumigatus, and C. neoformans.

Analysis of normal human sera (NHS) for the presence of chitin, modifiedchitin and laminarin antibodies outlined here demonstrate that theseantibodies are naturally acquired and exist in healthy individuals(FIGS. 14A through 14D). Chitin antibodies seem to be present atsignificant levels (FIG. 14A) and these levels are significantly higherthan those against β-glucan (FIG. 14D), It is interesting to note thatchitin antibodies are directed towards a conformational epitope made upof at least a saccharide of DP6 (FIG. 14C). These results are importantfor the design of a chitin-based vaccine because they suggest that anoptimum conjugate vaccine should contain chitin fragments of a least 6GlcNAc residues.

The vaccine produced as outlined in the preceding specification can beformulated in a pharmaceutically acceptable carrier, such as phosphatebuffered saline, normal saline or other appropriate carrier.Additionally, the vaccine can optimally include one or more adjuvants toaugment the immunogenecity and/or efficacy. Adjuvants can be addeddirectly to the vaccine compositions or can be administered separately,either concurrently with or shortly after, administration of thevaccine. Without limitation, suitable adjuvants include a variety ofadjuvants known in the art, either alone or in combination. Non-limitingexamples are an aluminum salt such as aluminum hydroxide gel or aluminumphosphate or alum, but can also be a salt of calcium, magnesium, iron orzinc, or may be an insoluble suspension of acylated tyrosine, oracylated sugars, cationically or anionically derivatized saccharides, orpolyphosphazenes. Adjuvants can also be selected, for example, from avariety of oil in water emulsions, Toll like receptors agonists, (exToll like receptor 2 agonist, Toll like receptor 3 agonist, Toll likereceptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8agonist and Toll like receptor 9 agonist), saponins or combinationsthereof.

A vaccine composition yielding a productive antibody response may beused in multiple ways. For example, the vaccine may be used for directimmunization of patients at risk for fungal infection. Alternatively,the compounds and compositions of the present invention may be used toisolate or generate antibodies to the compounds of the invention.Isolated antibodies might be prepared, either polyclonal or monoclonalin nature, using known methods available in the art. For example, thecompounds of the invention may be used to screen human antibody phagedisplay libraries, by methods well known to practitioners skilled in theart. As another example, the compositions described herein may be usedto elicit an antibody response in an appropriate host and the resultingantibodies may be immortalized by hybridoma technology, using knownmethods well established in the art. In yet another non-limitingexample, using methods known in the art, the compounds described hereinmay be used to directly produce human antibodies using human B-cellhybridoma technology. Any antibodies thus produced may be used to conferpassive protection, either for prophylactic or therapeutic use.Non-limiting examples for uses of these isolated antibodies mightinclude direct protection against fungal disease via immunotherapy,combination therapy with existing anti-fungal agents, or immunoconjugatepreparation, eg. direct conjugation to anti-fungal agents. Furthermore,these antibodies can be used to develop a diagnostic platform fordetection of fungal infection in patient samples.

In view of the described achievement in producing a vaccine antigen thatelicits specific responses to fungal cell wall components, any of theforegoing antibody based strategies could be extended to antibodyfragments or engineered antibody derivatives, which can include one ormore complementarity determining regions (CDRs) of antibodies, or one ormore antigen-binding fragments of an antibody. The terms “antibody” and“antibodies” include, but are not limited to, monoclonal antibodies,multispecific antibodies, human antibodies, humanized antibodies,camelised antibodies, chimeric antibodies, single-chain Fvs (scFv),single chain antibodies, single domain antibodies, Fab fragments, F(ab′)fragments, etc., and epitope-binding fragments of any of the above.

A portion of the chitin present in the fungal cell wall is modified bychitin deacetylases to produce chitosan and there is evidence that amixture of chitin/chitosan polymers may be necessary for proper cellwall integrity and function (19, 20). NMR data on the modified chitinfragments shows some residual free amino groups, raising the possibilitythat the vaccine can target both pure chitin as well as chitosancontaining regions in the chitinous cell wall.

In addition to chitin, β-mannan and β-glucan polymers comprise thefundamental carbohydrate constituents of the fungal cell wall. Vaccineinduced antibodies that target specific β-mannan or β-glucancarbohydrates have been shown to confer protection against fungaldisease, displaying both vaccine based efficacy and passive protection(9-11). To date, chitin has not been examined as an anti-fungal vaccinetarget. In vivo, these three carbohydrate components are covalentlycross linked to form the cell wall lattice (21, 22). Vaccineformulations consisting of modified chitin alone, or a combination oftwo or more of the conserved carbohydrate epitopes of the fungal cellwall, are contemplated to produce a broadly protective vaccine response.Such a combination vaccine could be designed either as a co-administeredmixture or as a cross linked scaffold that more closely mimics thesurface topography of the fungal cell wall. It is anticipated that thesevaccines will be highly effective at inducing a pan fungal immuneresponse.

The invention will be better understood by reference to the followingexamples, which illustrate but do not limit the invention describedherein.

EXAMPLES Example 1: Partial Re-N-Acetylation/Deamination of Chitosan toYield Modified Chitin Fragments

-   -   3 g of chitosan (16.8 mmol of GlcNH₂; Sigma cat. #419419) was        suspended in 150 mL of H₂O in a 250 mL round bottomed flask and        placed on a stir plate.    -   Acetic anhydride, 795 μL, 0.5 equivalents, relative to the        number of amino groups, was added to 15 mL of EtOH.    -   The acetic anhydride solution was added to the stirring chitosan        suspension, dropwise over 30 minutes, via an addition funnel.        The reaction became very viscous, but it was still possible to        stir the reaction mixture.    -   The reaction was allowed to proceed for another 30 minutes at        room temperature.    -   18 mL of glacial acetic acid was added to the reaction mixture,        giving a pH of ˜3.    -   6 mL of a freshly prepared 5% aqueous solution of NaNO₂ was        added to the reaction and the mixture was stirred for 1 hour at        room temperature. The reaction liberated a substantial amount of        gas and became much less viscous during the course of the        reaction.    -   The reaction mixture was neutralized with 16.5 mL of 10 N NaOH.    -   The sample was dialyzed overnight, against 4 L of 1 M NaCl, in a        dialysis membrane with a 7,000 molecular weight cut off. The        sample was further dialyzed against 4 changes of 4 L of water at        room temperature.    -   The reaction mixture was centrifuged for 15 minutes at 4,000 RPM        to remove the insoluble material, then filtered through 0.45 um        syringe filters.    -   Samples were removed for HPLC and NMR analysis.

Example 2: Preparation of Modified Chitin-Tetanus Toxoid VaccineConjugate by Reductive Amination

-   -   5 mg of tetanus toxoid (TT, 3.3 mL from 3 mg/mL solution in        saline; Sreum Staten Institute) was added to 50 mg of modified        chitin fragment.    -   The Schiff Base reaction was allowed to proceed for 6 hours at        room temperature.    -   Repurified sodium cyanoborohydride, 10 mg, was added in a volume        of 10 μL H₂O to the reaction mixture. The reaction was left to        proceed at room temperature and monitored after one and three        days by SDS-PAGE.    -   Following the reaction, the chitin-TT mixture was diluted to ˜2        mL with PBS and dialyzed against 4 L of PBS. The dialyzed sample        was filtered through a 0.45 μm syringe filter. The final        dialysate was quantitated by BCA assay and analyzed by SDS-PAGE        and HPLC.

Example 3: Immunization of Balb/C Mice with Modified Chitin-TT VaccineConjugate

40 female Balb/C mice were received and housed under standard day/nightcycles with food and water, ad libitum. The animals were allowed toacclimate to the facility for a minimum of one week, then randomlydivided into four groups and immunized as follows:

-   1. An emulsion of PBS in Freund's Complete Adjuvant (200 μL) was    delivered by intraperitoneal injection into 10 mice on day 0. On    days 28 and 38, animals received injections of PBS in Freund's    Incomplete Adjuvant, delivered in the same manner.-   2. Modified Chitin-TT conjugate (25 μg of antigen) was injected into    10 mice as an emulsion in Freund's Complete Adjuvant (200 μL), by    intraperitoneal injection on Day 0. On days 28 and 38, animals    received booster injections of Chitin-TT (25 μg) in Freund's    Incomplete Adjuvant, delivered in the same manner as the primary    immunization.-   3. Animals were injected with Modified Chitin-TT (50 μg), as per the    protocol for Group 2.-   4. Animals were injected with Modified Chitin-TT (100 μg), as per    Groups 2 & 3.    -   Mice were bled and serum was prepared on Days −4, 38 and 50 in        order to evaluate chitin specific antibody responses.

Animals were bled and serum was prepared 4 days prior to the initialinjection (Study Day −4), in order to determine antibody titers inpre-immune serum. Mice were immunized on Study Day 0 according to theexperimental groups outlined above. The LMW Chitin-TT antigen wassuspended in PBS at concentrations of 0.25, 0.5 and 1.0 mg/mL, mixedwith Freund's Complete adjuvant at a 1:1 ratio, and vortexed for 20minutes to create an emulsion. 200 μL of emulsion containingPBS/Freund's control or the test antigens (final conc. 0.125, 0.25 or0.5 mg/mL for a total of 25, 50, or 100 μg protein conjugate,respectively) was administered by intraperitoneal injection, asindicated above, to each mouse on Day 0. For the subsequent boosts onDay 28 and Day 38, antigen was prepared similarly in Freund's IncompleteAdjuvant and administered in the same manner. Immunized animals werebled and serum was prepared on Day 38 and Day 50 in order to evaluateantibody responses.

Example 4: Assay for Antibody Response of Balb/C Mice Immunized withModified Chitin-TT Vaccine Conjugate

-   -   2 μg/mL solutions of two different Modified Chitin human serum        albumin (HSA) conjugates were prepared in PBS.    -   100 μL of each solution was coated into half the wells of a        96-well assay plate (½ plate for each antigen) and incubated        overnight at room temperature.    -   The wells were washed 3× with PBST, then blocked for 1 hour with        1% BSA in PBS.    -   Wells were washed 3× with PBST.    -   Modified Chitin Vaccine mouse sera were pooled and diluted 1:20        in PBS.    -   Treatment groups were as follows:        -   1. PBS/Freund's        -   2. Modified Chitin-TT/Freund's; 25 μg/mouse        -   3. Modified Chitin-TT/Freund's; 50 μg/mouse        -   4. Modified Chitin-TT/Freund's; 100 μg/mouse    -   200 μL of a ten fold dilution of each pooled serum sample was        plated into the first column of wells of the Chitin-HSA coated        microplate.    -   A two fold dilution series was made for a total of 12 different        serum concentrations, ranging from 1:200 to 1:409,600.    -   The samples were incubated for 2 hours at room temperature.    -   The plate was washed 3× with PBST, then incubated 1 hour at RT        with 100 μL of anti-mouse IgG (γ-chain specific)-HRP diluted        1:2500 in 1% BSA/PBST    -   Wells were washed 3× with PBST, then incubated with 100 μL of        SureBlue Reserve TMB peroxidase substrate for 5 minutes at room        temperature.    -   The reaction was stopped with 100 μL 1 N HCl.    -   The absorbance was read on a microplate reader at 450 nm.

Example 5: Assay for Specificity of the Antibody Response of Balb/C MiceImmunized with Modified Chitin-TT Vaccine Conjugate A. AntigenInhibition

-   -   2 μg/mL solutions of modified chitin HSA conjugates (lots #2 and        #3) were prepared in PBS.    -   100 μL of each solution was coated into half the wells of a        96-well assay plate and incubated overnight at room temperature.    -   The wells were washed 3× with PBST and blocked 1 hour with 1%        BSA in PBS.    -   Wells were washed 3× with PBST and stored dry at 4° C. until        use.    -   2 mg/mL solutions of GlcNAc, HA and modified Chitin fragments        were prepared in PBS and 200 μL of each solution was transferred        to a set of titer tubes.    -   A two fold dilution series in PBS was made for a total of 11        different concentrations of each inhibitor, ranging from 2 mg/mL        to 1.95 μg/mL, as well as an uninhibited control (final        inhibitor concentrations of 1 mg/mL to 976 ng/mL).    -   Pooled sample from the Modified Chitin-TT/Freund's treatment        group (100 μg/mouse, Day 50 bleed) was diluted to 20:000 fold in        1% BSA/PBST.    -   100 μL of diluted serum sample was transferred into each of the        titer tubes (final 40:000 dilution) and incubated with the        inhibitors for 30 minutes at room temperature.    -   100 μL of each serum sample/inhibitor was transferred into the        Modified Chitin-HSA coated microplate and incubated for 2 hours        at room temperature.    -   The plate was washed 3× with PBST, then incubated 1 hour at RT        with 100 μL of anti-mouse IgG (γ-chain specific)-HRP diluted        1:2500 in 1% BSA/PBST    -   Wells were washed 3× with PBST, then incubated with 100 μL of        SureBlue Reserve TMB peroxidase substrate for 5 minutes at room        temperature.    -   The reaction was stopped with 100 μL 1 N HCl and the absorbance        was read on a microplate reader at 450 nm.

B. Antibody Reactivity Specificity Controls

-   -   2 μg/mL solutions of the test compounds (serine-O-GlcNAc,        ovalbumin, fetuin, hyaluronic acid (HA), fetal bovine serum)        were prepared in PBS, with the exception of FBS, which was        prepared as a 10% solution (v:v) in PBS.    -   100 μL of each solution was coated into the wells of a 96-well        assay plate. The test antigens were incubated in the plate        overnight at 4° C.    -   The wells were washed 3× with PBS and blocked for 1 hour with 1%        BSA/PBS.    -   Wells were washed 3× with PBST and stored dry at 4° C. until        use.    -   Modified Chitin Vaccine mouse sera were pooled by treatment        group and diluted 1:20 in PBS Treatment groups were as follows:        -   1. PBS/Freund's        -   2. Modified Chitin-TT/Freund's; 25 μg/mouse        -   3. Modified Chitin-TT/Freund's; 50 μg/mouse        -   4. Modified Chitin-TT/Freund's; 100 μg/mouse    -   The pooled serum samples were further diluted to a final of        1:20,000 fold in 1% BSA/PBST.    -   100 μL of diluted serum sample from each group was transferred        in triplicate to the wells of the assay plate and incubated for        1 hour at room temperature.    -   The plate was washed 3× with PBST, then incubated 1 hour at RT        with 100 μL of anti-mouse IgG (γ-chain specific)-HRP diluted        1:2500 in 1% BSA/PBST    -   Wells were washed 3× with PBST, then incubated with 100 μL of        SureBlue Reserve TMB peroxidase substrate for 5 minutes at room        temperature.    -   The reaction was stopped with 100 μL 1 N HCl.    -   The absorbance was read on a plate reader at 450 nm.    -   Note that the ELISA response for all of the replicates for Group        4 were out of the linear range. These samples were arbitrarily        assigned a value of 3.0, which is near the detection limit of        the plate reader. These values probably represent an        underestimate.

C. Chitin/Chitosan Absorption and Inhibition

-   -   2 μg/mL solutions of the test compounds (modified chitin-HSA or        LMW HA-HSA) were prepared in PBS.    -   100 μL of each solution was coated into the wells of a 96-well        assay plate and incubated overnight at 4° C.    -   The wells were washed 3× with PBST and blocked for 1 hour with        1% BSA in PBS.    -   Wells were washed 3× with PBST.    -   In the meantime, pooled Modified Chitin Vaccine mouse sera was        diluted to a final concentration of 1:5000 in 1% BSA/PBS.    -   HA MAb #2 was diluted to 1:2000 in 1% BSA/PBS.    -   5 mL of diluted serum sample or HA MAb was incubated overnight        on a rotator at 4° C. with 100 mg of either chitin or chitosan        (both made as insoluble suspensions).    -   A fraction of diluted chitin vaccine #1 serum or HA MAb was set        aside at 4° C. to serve as positive controls and to mix with        chitin/chitosan extracts for inhibition experiment.    -   In parallel, chitin and chitosan was incubated with 5 mL of 1%        BSA/PBS in the same manner, in order to extract soluble        chitin/chitosan for inhibition testing.    -   Samples were centrifuged 5 minutes at 4500 to pellet out        insoluble chitin and chitosan.    -   The supernatant fractions were removed to fresh tubes and        diluted 4 fold (final 1:20,000 for Modified Chitin Vaccine and        1:8,000 for HA MAb) in 1% BSA/PBS.    -   Untreated Modified Chitin vaccine serum sample and untreated HA        MAb were likewise diluted to equivalent final concentrations in        1% BSA/PBS (100 μL Ab or serum+300 μL BSA/PBS)    -   For inhibition testing, untreated serum or Ab solutions were        diluted 4 fold with either chitin or chitosan extract to        equivalent final concentrations (final 1:20,000 for Modified        Chitin Vaccine and 1:8,000 for HA MAb). The solutions were        pre-incubated for 30 minutes at room temperature prior to        addition to the ELISA plate.    -   100 μL of each sample was added, in triplicate, to ELISA strips        coated with either modified Modified Chitin-HSA or LMW HA-HSA,        as appropriate.    -   The samples were incubated in the strips for one hour at room        temperature.    -   The strips were washed 3× with PBST, then incubated 1 hour at RT        with 100 μL of anti-mouse IgG (γ-chain specific)-HRP, diluted        1:2500 in 1% BSA/PBST.    -   Wells were washed 3× with PBST, then incubated with 100 μL of        SureBlue Reserve TMB peroxidase substrate for 5 minutes at room        temperature.    -   The reaction was stopped with 100 μL 1 N HCl.    -   The HA ELISA samples developed color very rapidly, so the        reaction was stopped within ˜1 minute. Likewise, the positive        control in the Modified Chitin Vaccine samples developed        rapidly, so the reaction was terminated after 2 minutes.    -   The absorbance was read on a microplate reader at 450 nm.

Example 6: Assays for Immunoreactivity of Serum Antibodies from ModifiedChitin-TT Vaccine Immunized Balb/C Mice Toward C. albicans Whole Cells

A. Binding of whole serum to C. albicans cells

-   -   A single colony of C. albicans was picked from a Saboraud        Dextrose Agar plate and used to inoculate 2 mL of YPD growth        medium. The culture was grown overnight at 30° C., with shaking        at 250 RPM.    -   100 μL of a 5×10⁶ cells/mL suspension was added into each well        of an amine surface modified Strip Well microplate. The cells        were allowed to settle for one hour at room temperature.    -   100 μL of a 2% solution of glutaraldehyde in PBS was added to        each well.    -   The cells were allowed to cross link overnight at room        temperature.    -   The fixative was aspirated away and the wells were washed 3×        with PBST.    -   Wells were blocked for 1 hour at room temp with 1% BSA in PBS.    -   In the meantime, pooled Modified Chitin Vaccine mouse sera were        diluted to a concentration of 1:20,000 in 1% BSA/PBS.    -   100 μL of 2 mg/mL solutions of modified chitin or PBS were        distributed, in triplicate, into titer tubes.    -   For inhibition testing, 100 μL of serum was added to the tubes        containing the inhibitors (final 1:40,000 for Modified Chitin        Vaccine serum samples and 1 mg/mL for inhibitors). The solutions        were pre-incubated for 30 minutes at room temperature prior to        addition to the ELISA plate.    -   100 μL of each sample was added to the C. albicans coated        stripwell plate. The samples were incubated for one hour at room        temperature.    -   The strips were washed 3× with PBST, then incubated 1 hour at RT        with 100 μL of anti-mouse IgG (γ-chain specific)-HRP, diluted        1:2500 in 1% BSA/PBST.    -   Wells were washed 3× with PBST, then incubated with 100 μL of        SureBlue Reserve TMB peroxidase substrate at room temperature.    -   The reaction was stopped after two minutes with 100 μL 1 N HCl.    -   The absorbance was read on a plate reader at 450 nm.        B. Binding of Affinity Purified Modified Chitin Reactive        Antibodies to C. albicans    -   20 mg of modified chitin and periodate oxidized HA were        dissolved in 2 mL of 20 mM NaPO₄, pH 7.5. Oxidized HA required        overnight at room temperature to dissolve.    -   UltraLink Hydrazide gel (4 mL of slurry, 2 mL of settled resin)        was washed with 5 volumes of 20 mM NaPO₄, pH 7.5.    -   The dissolved saccharides were added to the washed resin and        allowed to react for 24 hours at room temperature.    -   The unbound material was drained away and the resins were washed        with the following:        -   1. 20 column volumes of 20 mM NaPO₄, pH 7.5        -   2. 5 column volumes of 10×PBS.        -   3. 10 column volumes of 1×PBS.    -   Pooled Modified Chitin Vaccine mouse sera (from PBS control        group and 100 μg modified chitin-TT immunized group) were        diluted to an initial concentration of 1:40 in 1% BSA/PBS    -   100 μL (200 μL of slurry) of Modified Chitin and oxidized HA        UltraLink hydrazide gels were washed with 500 μL of PBS in a        micro spin column.    -   200 μL of each serum and the affinity resins were mixed and        incubated for 1 hr at RT.    -   The unbound material was removed by centrifugation for 30        seconds at 8500 RPM.    -   The resins were washed 3× with 500 μL PBST.    -   200 μL of 0.2 M glycine, pH 2.8 was added to each resin and        allowed to interact for ˜3 min.    -   Eluted protein was collected by centrifugation directly into a        tube containing 50 μL of 1 M Tris, pH 8.    -   A single colony of C. albicans was picked from a Sabouraud        Dextrose Agar plate and used to inoculate 2 mL of YPD growth        medium and the culture was grown overnight at 30° C., with        shaking at 250 RPM.    -   100 μL of 1×10⁷ cells/mL suspension was added into each well of        an amine surface modified Strip Well microplate. The cells were        allowed to settle for 2 hours at room temperature.    -   100 μL of a 2% solution of glutaraldehyde in PBS was added to        each well.    -   The cells were allowed to cross link for one hour at room        temperature.    -   The fixative was carefully aspirated away and the wells were        washed 3× with PBST.    -   Wells were blocked for 1 hour at room temp with 1% BSA in PBS.    -   The antigen affinity purified antibodies were diluted 1:1,000        with 1% BSA in PBS.    -   A 2 fold dilution series was prepared in titer tubes.    -   100 μL of the antibody dilutions were added in triplicate to        the C. albicans coated plate.    -   The samples were incubated for 1 hour at room temperature.    -   The plate was washed 3× with PBST, then incubated 1 hour at RT        with 100 μL of anti-mouse IgG (γ-chain specific)-HRP, diluted        1:2500 in 1% BSA/PBST.    -   Wells were washed 3× with PBST, then incubated with 100 μL of        SureBlue Reserve TMB peroxidase substrate for 5 minutes at room        temperature.    -   The reaction was stopped with 100 μL 1 N HCl.    -   The absorbance was read on a plate reader at 450 nm.        C. Binding of Vaccine-Induced Antibodies to Candida albicans by        Flow Cytometry:

Candida Cells:

Using a sterile loop, C. albicans from frozen stock were streaked on anSDA plate and incubated at 30 C. A single colony from the SDA plate waspicked using inoculating loop and inoculated into a 250-ml flaskcontaining 50 ml of YPD and incubated at 30 C with shaking at 150 rpmfor at least 18 h. For experiments where mycelia stage of candida wasused, the cells were incubated at 37 C for 150 minutes and 300 minutesin YPD containing serum following O/N incubation to induce and favordifferentiation into mycelia stage of candida. The O/N culture wastransferred into a 50 ml tube and centrifuged at 1000 g for 20 mins andthe pellet was washed with 2 ml PBS solution at least 3 times. Thepellet was resuspended in 2 ml PBS and the concentration was adjusted to0.1 OD600 (4×106/ml).

Cryptococcal Cells:

Fungal growth-streaking from stock overnight was performed using asterile loop, and streaking the strain Cryptococcus neoformans type Afrozen stock to isolation on an SDA plate and incubated at 30° C. Asingle colony from the SDA plate was picked using inoculating loop andinoculated into a 250-ml flask containing 50 ml of YPD and incubated at30 C with shaking at 150 rpm for at least 18 h. For experiments wheremycelia stage of Cryptococcus was used, the cells were incubated at 37 Cfor 150 minutes and 300 minutes in YPD containing serum following O/Nincubation to induce and favor differentiation into mycelia stage ofcandida. The O/N culture was transferred into a 50 ml tube andcentrifuged at 1000 g for 20 mins and the pellet was washed with 2 mlPBS solution at least 3 times. The pellet was resuspended in 2 ml PBSand the concentration was adjusted to 1.0 OD600 reading (4×10 7/ml).

Sera and Antibodies:

Mice sera from various vaccine groups was diluted in 1:10, 1:50 and1:100 in PBS and used at 15 ul volume/tube. The control antibodies werediluted as: Mouse mAb to candida, 1:5, 1:10 and 1:20 dilutions; ForRabbit pAb to candida, 1:50, 1:100 and 1:200 dilutions and used at 20 ulvolume/tube. FITC-labeled secondary antibodies were diluted 1:25 in PBSand used at 15 ul/tube. Propidium Iodide was used at 1:2 in PBS (5ul/tube).

Flow Cytometry:

About 100 ul of fungal cells were transferred into the tubes andappropriate serum and antibodies were added and incubated on ice for 60minutes. The cells were then washed 2 times with 1 ml PBS (By spinningat 1000 g for 10 mins at 40 C, decanting supernatant) followed byincubation with secondary antibody for 30 mins on ice protected fromlight.

Diluted propidium iodide (No Wash) was added and the cells incubated foran additional 10 mins on ice followed by 2 PBS washes and finally fixedin 200 ul of 1% paraformaldehyde. The data was then acquired on a FACSCalibur instrument.

Example 7: Modified Chitin-TT Vaccine Confers Protection from a LethalChallenge of C. albicans Pathogenic Fungus

A. Immunization of Balb/C Mice with Modified Chitin-TT Vaccine, and CD1Mice with Chitoheaose-TT and Laminarin-TT Conjugates.

-   -   40 female mice were received and housed under standard day/night        cycles with food and water, ad libitum. The animals were allowed        to acclimate to the facility for a minimum of one week, then        randomly divided into two groups and immunized as follows:    -   Initially, BalB/c mice were divided into two groups, n=20        mice/group:        -   1. PBS/Freund's; 200 μL/mouse; intraperitoneal injection        -   2. Mod Chitin-TT/Freund's; 200 μL/mouse; i.p.; 100 μg/mouse    -   For the mock immunized control animals, an emulsion of PBS in        Complete Freund's Adjuvant (200 μL) was delivered by        intraperitoneal injection into 20 mice on day 0. On days 28 and        38, animals received injections of PBS in Incomplete Freund's        Adjuvant, delivered in the same manner as the primary        immunization.    -   For the vaccination group, Modified Chitin-TT conjugate (100 μg        of antigen, as an emulsion in Complete Freund's Adjuvant) was        delivered by intraperitoneal injection into 20 mice (200 μL), on        Day 0. On days 28 and 38, animals received booster injections of        Chitin-TT (50 μg) in Incomplete Freund's Adjuvant, delivered in        the same manner as the primary immunization.    -   Mice were bled and serum was prepared on Days −4, 38, 50, and        62, for evaluation of antibody responses.    -   Same protocol was used to immunize CD1 mice with modified        chitin-TT; laminarin-TT and Chitohexaose-TT conjugates        B. Challenge of Chitin-TT Vaccine Immunized Balb/C and CD1 Mice        with a Lethal Dose of C. albicans

The animals from each of the immunization groups, above, were randomlydivided into three groups of ten for each immunization cohort, andinoculated with C. albicans cells, as follows.

Experimental groups: n=10 mice/group, randomly divided into groups

Groups 1 and 2 comprise animals from PBS (mock) immunized animals

-   -   1. Saline; 200 mouse; tail vein injection    -   2. 5×10⁶ C. albicans; 200 μL from 2.5×10⁷ CFU/mL; tail vein        injection Groups 3 and 4 comprise animals from modified chitin        TT immunized animals    -   3. Saline; 200 μL/mouse; tail vein injection    -   4. 5×10⁶ C. albicans; 200 μL from 2.5×10⁷ CFU/mL; tail vein        injection

Monitoring Mice:

-   -   1. Mice were checked daily for mortality and signs of distress:        -   Decreased food and water consumption, weight loss,            self-imposed isolation/hiding, rapid breathing,            opened-mouthbreathing, increased/decreased movement,            abnormal posture/positioning, dehydration, twitching,            trembling, and tremors.    -   2. Mice showing signs of distress accompanied by any of the        following were defined as moribund: impaired ambulation (unable        to reach food and water), evidence of muscle atrophy or signs of        emaciation, lethargy (drowsiness, aversion to activity, lack of        physical or mental alertness), prolonged anorexia, difficulty        breathing, and neurological disturbances. Mice were euthanized        when they look moribund and death appeared imminent. The date of        death was recorded as the following day.

Example 8: Detection and Characterization of Chitin and β-GlucanAntibodies in Human Normal Sera

2 μg/mL solutions of different human serum albumin (HSA) conjugates(modified chitin, chitohexaose, and laminarin) were prepared in PBS.

-   -   100 μL of each solution was coated into half the wells of a        96-well assay plate (½ plate for each antigen) and incubated        overnight at room temperature.    -   The wells were washed 3× with PBST, then blocked for 1 hour with        1% BSA in PBS.    -   Wells were washed 3× with PBST.    -   Normal human sera diluted 1:20 in PBS.    -   200 μL of a ten fold dilution of each pooled serum sample was        plated into the first column of wells of the various HSA        conjugates coated microplate.    -   A two fold dilution series was made for a total of 12 different        serum concentrations, ranging from 1:200 to 1:409,600.    -   The samples were incubated for 2 hours at room temperature.    -   The plate was washed 3× with PBST, then incubated 1 hour at RT        with 100 μL of anti-human IgG (γ-chain specific)-HRP diluted        1:2500 in 1% BSA/PBST    -   Wells were washed 3× with PBST, then incubated with 100 μL of        SureBlue Reserve TMB peroxidase substrate for 5 minutes at room        temperature.    -   The reaction was stopped with 100 μL 1 N HCl.    -   The absorbance was read on a microplate reader at 450 nm.

Example 9: Preparation of a β1,3 Glucan/Chitin Conjugate Tetanus ToxoidVaccine

Following is an example of one of several envisioned methods to preparea combination fungal vaccine comprising two or more conserved componentsof the fungal cell wall. In this example, laminarin (linear, unbranchedpolymer of β-1,3 glucan) was reduced and oxidized with sodium periodate,subsequently conjugated directly to chitosan via reductive amination,and finally, the cross linked carbohydrates were conjugated to tetanustoxoid.

-   -   Laminarin was reduced with sodium borohydride and subsequently        oxidized with sodium meta periodate by standard methods.    -   100 mg of oxidized laminarin was dissolved in PBS (several        minutes at 37° C. to dissolve) and mixed with low molecular        weight chitosan at a 1:5 ratio. The chitosan remained as an        insoluble suspension.    -   The reaction was placed in a 37° C. shaking incubator for 24        hours.    -   20 mg of NaCNBH₃ was added and the reaction was continued        another 24 hours at 37° C.    -   The reaction suspension was transferred to a disposable plastic        column and the insoluble material was washed extensively with        H₂O to remove any residual, unreacted laminarin.    -   The washed, insoluble material was resuspended in 5 mL of 10%        aqueous acetic acid    -   1 mL of freshly prepared aqueous sodium nitrite was added and        the reaction was allowed to proceed for 60 minutes at room        temperature. Most of the insoluble material entered into        solution during the course of the reaction.    -   The reaction mixture was centrifuged 5 minutes at 4500×G to        remove the remaining insoluble material. The soluble fraction        was further processed as described below.    -   The soluble fraction was diluted to 15 mL, in a final        concentration of 1 M NaCl and dialyzed against 4 changes of 4        liters of H₂O. The dialyzed material was lyophilized to give a        fluffy white powder, 90 mg total yield.    -   The polysaccharide conjugate was analyzed by SEC HPLC and the        presence of both chitin and β-glucan in the product was        confirmed by ¹H NMR.    -   10 mg of tetanus toxoid (TT, 3.3 mL from 3 mg/mL solution in        saline) was added to 40 mg of the laminarin-chitin conjugate.    -   A 2 μL aliquot of the sample was added to 98 μL of 1×LDS sample        buffer+5 μL of 1 M DTT for SDS-PAGE analysis.    -   Another 2 μL of sample was added to 98 μL of 1×PBS for HPLC        analysis.    -   The solution was allowed to react for 24 hours at 37° C. in a        shaking incubator (300 RPM).    -   Repurified sodium cyanoborohydride, 40 mg, was added directly to        the reaction. The reactions were left to proceed for 24 hours at        37° C. in a shaking incubator (300 RPM).    -   Following the reaction, the conjugation reaction was        concentrated to ˜2.5 mL in an Amicon ultrafiltration device with        a 10,000 MWCO membrane. The sample was dialyzed against 3        exchanges of 4 L of PBS. The final dialysate was quantitated by        BCA assay and analyzed by SDS-PAGE and SEC HPLC. After the        samples were adjusted to a concentration of 1 mg/mL, they were        sterile filtered through a 0.22 micron filter.

LITERATURE REFERENCES

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What is claimed is:
 1. A compound comprising one or more polysaccharidemoieties each independently represented by the formulaβ(1→4)-[GlcNH-R]_(n)-2,5-anhydromannose, wherein n is a positive integerfrom 3 to 500, R is H or an acyl group, and the one or morepolysaccharide moieties are chitin/chitosan mied polymers; and animmunogenic carrier protein covalently linked to the one or morepolysaccharide moieties at the anhydromannose group of thepolysaccharide moieties.
 2. The compound of claim 1, wherein n is apositive integer from 3 to
 100. 3. The compound of claim 2, wherein n isa positive integer from 6 to
 50. 4. The compound of claim 1, wherein theacyl group R is an acetyl group.
 5. The compound of claim 1, wherein atleast 30% of the acyl groups in the compound are acetyl.
 6. The compoundof claim 1, wherein the carrier protein is tetanus toxoid.
 7. Thecompound of claim 1, wherein the carrier protein is diphtheria toxoid.8. The compound of claim 1, wherein the carrier protein is a fungalprotein virulence factor.
 9. The compound of claim 1, further comprisingone or more linear β-mannan or glucan epitopes covalently linked to thecarrier protein.
 10. The compound of claim 9, wherein the β-mannan is aβ-1,2-mannose tetrasaccharide.
 11. The compound of claim 9, wherein oneof the one or more glucan epitopes is a linear α-1,3-glucan epitope. 12.The compound of claim 9, wherein one of the one or more glucan epitopesis a linear β-1,3-glucan epitope.
 13. The compound of claim 1, whereinthe carrier protein is covalently linked to the one or morepolysaccharide moieties via one or more immunogenic scaffold moieties.14. The compound of claim 13, wherein each of the scaffold moieties isselected from the group consisting of a polysaccharide, a peptidoglycan,a polypeptide, and a polyglycerol.
 15. The compound of claim 13, whereinthe scaffold is linear.
 16. The compound of claim 13, wherein thescaffold is dendrimeric.
 17. The compound of claim 13, furthercomprising one or more linear β-mannan or glucan epitopes covalentlylinked to the carrier protein or the scaffold.
 18. The compound of claim17, wherein the β-mannan is a β-1,2-mannose tetrasaccharide.
 19. Thecompound of claim 17, wherein one of the one or more glucan epitopes isa linear α-1,3-glucan epitope.
 20. The compound of claim 17, wherein oneof the one or more glucan epitopes is a linear β-1,3-glucan epitope. 21.A composition comprising the compound of claim 1, wherein for at least80% of molecules of the compound in the composition n has a value from 6to
 50. 22. A composition comprising the compound of claim 1, wherein themean value of n is from 10 to 50.